A selection of important recent references
on Coelenterazine and Luciferases which utilize Coelenterazine

Authors
Shimomura O. Johnson FH.

Title
Peroxidized coelenterazine, the active group in the photoprotein aequorin.

Source
Proceedings of the National Academy of Sciences of the United States of America. 75(6):2611-5, 1978 Jun.

Abstract
The photoprotein aequorin emits light by an intramolecular reaction when Ca2+ is added under either aerobic or anaerobic conditions. Previously reported evidence has indicated two possibilities: (i) the functional group of aequorin is coelenterazine itself, a compond that plays key roles in the bioluminescence of various other types of organisms, or (ii) it is the enolized form of this compound. Present data rule out both of these possibilities, through elucidation of the structure of the yellow compound that is split off aequorin by treatment with NaHSO3. The yellow compound is now shown to be a tertiary alcohol of coelenterazine on the basis of chemical reactions, mass spectral data, and relationships to known derivatives of coelenterazine. From this structure and the method of forming the yellow compound from aequorin, aequorin evidently contains a peroxide of coelenterazine as the active group. The presence of such a peroxide is consistent with the fact that aequorin yields free coelenterazine upon treatment with Na2S2O4. Although there is no applicable technique at present to determine with assurance the specific state of the peroxide in the protein, a study with 18O tracer indicates that a linear peroxide structure is more likely than the alternative possibility of a dioxetane structure.

Authors
Rutter GA. White MR. Tavare JM.

Institution
Department of Biochemistry, School of Medical Sciences, University of Bristol, UK.

Title
Involvement of MAP kinase in insulin signalling revealed by non-invasive imaging of luciferase gene expression in single living cells.

Source
Current Biology. 5(8):890-9, 1995 Aug 1.

Abstract
BACKGROUND: Studies of the mechanisms by which signals are transmitted from receptor tyrosine kinases would be facilitated by a way of monitoring events at the single-cell level. We have explored how luciferase imaging can be used to examine the role of specific signalling pathways in insulin-stimulated gene expression. The analysis of luciferase expression in single cells has previously been hampered by the insensitivity of existing methodologies and the lack of a way of monitoring quantitatively, and independently, more than one promoter within the same cell. We have developed a technique for examining the dynamics of insulin-stimulated AP-1-dependent transcription in single living cells, and have explored the signalling pathway involved. RESULTS: Luciferase and aequorin gene expression were examined in single living cells with a high-sensitivity photon-counting camera. The technique involved the comicroinjection of luciferase- and aequorin-based reporter plasmids directly into the cell nucleus, and the subsequent analysis of luminescence in the presence of luciferin and coelenterazine, respectively. The method is quantitative and allows insulin-stimulated gene expression to be monitored in real time. We found that insulin promoted a substantial increase in the expression of a luciferase gene under the control of the AP-1-binding site from the collagenase gene promoter. Aequorin expression, under the control of a cytomegalovirus promoter, was unaffected by insulin. The effect of insulin on luciferase expression was specifically blocked by overexpression of either the mitogen-activated protein (MAP) kinase phosphatase CL100, or the dominant-negative mutant MAP kinase kinase, MEKS217/221A. CONCLUSIONS: Microinjection coupled with luciferase imaging allows hormone-regulated gene expression from relatively weak promoters to be monitored in single living cells. We have used this method to demonstrate that MAP kinase plays a central role in the ability of insulin to stimulate AP-1-dependent gene transcription.

Authors
Shimomura O.

Institution
Marine Biological Laboratory, Woods Hole, MA 02543.

Title
Cause of spectral variation in the luminescence of semisynthetic aequorins.

Source
Biochemical Journal. 306 ( Pt 2):537-43, 1995 Mar 1.

Abstract
Aequorin emits light in the presence of Ca2+, decomposing into apoaequorin, coelenteramide and CO2. Semisynthetic aequorins, produced by replacing the coelenterazine moiety in aequorin with analogues of coelenterazine, showed widely different sensitivities to Ca2+ as well as certain spectral variations. A group of semisynthetic aequorins, e-type aequorins, showed bimodal luminescence, with peaks at 400-405 nm and 440-475 nm in various intensity ratios, whereas all other aequorins luminesced with only one peak, in the range 440-475 nm. The cause of the spectral variation was studied by various experiments including: (1) comparison with the fluorescence of the spent solution and the luminescence of the spent solution produced by added coelenterazine; (2) luminescence in 2H2O; (3) the rate of conformational change of apoaequorin; (4) the rates of regeneration in the presence and absence of O2. The results suggested that the spectrum of Ca(2+)-triggered luminescence is strongly affected by the ionic charge on the amide N atom of the coelenteramide that is bound to apoaequorin. When the amide N atom is negatively charged, light is emitted with a 440-475 nm peak. In the case of e-type aequorins, the negative charge on the amide N atom is less because of the structure of e-coelenterazine involved, resulting in the emission of a 400-405 nm peak from the uncharged form of coelenteramide; the intensity ratio of 400-405 nm peak to 440-475 nm peak is determined by the amount of negative charge resting on the amide N atom of e-coelenteramide at the time of light emission. Most of the spectral variations in luminescence and fluorescence can be explained on the basis of ionic and hydrophobic interaction between a coelenteramide and apoaequorin.

Authors
Badminton MN. Kendall JM. Sala-Newby G. Campbell AK.

Institution
Department of Medical Biochemistry, University of Wales College of Medicine, Cardiff, United Kingdom.

Title
Nucleoplasmin-targeted aequorin provides evidence for a nuclear calcium barrier.

Source
Experimental Cell Research. 216(1):236-43, 1995 Jan.

Abstract
The Ca(2+)-activated photoprotein aequorin has been targeted to the nucleus of live cells by engineering nucleoplasmin, a nuclear structural protein from Xenopus laevis, onto the amino terminus. Successful targeting of the apoprotein was demonstrated by immunolocalization and selective permeabilization of the plasma membrane. Functional protein was reconstituted in live cells by incubation with coelenterazine. The effect of elevating cytosolic Ca2+ on nuclear Ca2+ was investigated in populations of live COS7 cells expressing either cytosolic aequorin or nuclear aequorin. Incubation of cells with ionomycin, in the absence of external Ca2+, released Ca2+ from internal stores causing an increase in chemiluminescent light emission from cytosolic aequorin but not nuclear aequorin. Nonagonist-dependent movement of Ca2+ across the plasma membrane, induced by the membrane attack complex of complement, causes a large increase in cytosolic Ca2+ which triggered 95% of cytosolic aequorin but only 50-60% of nuclear aequorin. These results provide clear evidence for a nucleocytoplasmic barrier to Ca2+.

Authors
Kendall JM. Badminton MN. Dormer RL. Campbell AK.

Institution
Department of Medical Biochemistry, University of Wales College of Medicine, Cardiff, United Kingdom.

Title
Changes in free calcium in the endoplasmic reticulum of living cells detected using targeted aequorin.

Source
Analytical Biochemistry. 221(1):173-81, 1994 Aug 15.

Abstract
The Ca(2+)-activated photoprotein aequorin has been engineered with the endoplasmic reticulum (ER)-targeting sequence from calreticulin at the N-terminus and the KDEL sequence at the C-terminus so that it locates in the ER of living cells. Targeting of apoaequorin to the ER of COS7 cells was demonstrated by immunolocalization. Selective permeabilization of cells expressing the modified protein suggested that targeting was highly efficient. Functional photoprotein was reconstituted in live cells by incubating them with coelenterazine. Light emission from cells expressing ER aequorin showed that the estimated free Ca2+ within the ER of live cells at 37 degrees C was 0.3-1.0 microM, some 10 times that in the cytosol. An increase in the rate constant for aequorin light emission was demonstrated when the cells were warmed from 4 degrees C. This increase could be in part, but not wholly, explained by an increase in rate constants for aequorin at higher temperatures and a change in kinetics as a result of the ER targeting of aequorin. The increase in rate constants in the cells was inhibited by thapsigargin and occurred in the presence or absence of extracellular Ca2+. These results highlight the importance of converting aequorin light emission to rate constants and of calibrating any variants if qualitative and quantitative conclusions are to be drawn about free Ca2+ in intracellular compartments.

Authors
Saran S. Nakao H. Tasaka M. Iida H. Tsuji FI. Nanjundiah V. Takeuchi I.

Institution
National Institute for Basic Biology, Okazaki, Japan.

Title
Intracellular free calcium level and its response to cAMP stimulation in developing Dictyostelium cells transformed with jellyfish apoaequorin cDNA.

Source
FEBS Letters. 337(1):43-7, 1994 Jan 3.

Abstract
A new method is described for measuring intracellular free calcium concentrations, [(Ca2+)i], in the cells of Dictyostelium discoideum transformed with apoaequorin cDNA of the jellyfish, Aequorea victoria. Aequorin, a calcium-specific indicator, was regenerated in vivo from apoaequorin produced in the cells by incubation with coelenterazine. The results showed that [(Ca2+)i] in developing cells markedly increases at the aggregation stage and again at the culmination stage after a temporary drop at the migration stage. Except for the vegetative stage, the cells at all stages of development exhibit a sharp transient increase in [(Ca2+)i] upon stimulation with a cAMP (50 nM) pulse, high responses being observed at the migration and culmination stages. Separated prestalk cells of migrating slugs contain more than twice as much [(Ca2+)i] and show three times as large a response to cAMP stimulation as prespore cells.

Authors
Button D. Brownstein M.

Institution
Laboratory of Cell Biology, NIMH, NIH, Bethesda, Maryland.

Title
Aequorin-expressing mammalian cell lines used to report Ca2+ mobilization.

Source
Cell Calcium. 14(9):663-71, 1993 Oct.

Abstract
Mammalian cells that stably express jellyfish aequorin have been used to report activation of Ca2+ mobilization by cell-surface receptors. Expression of aequorin cDNA (pAEQ) was driven by the cytomegalovirus (CMV) promoter in CHO-K1 and 293 cells. Clonal isolates were obtained which express high levels of apo-aequorin protein, the Ca(2+)-dependent luminescence of which is generated by treatment of living cells with the coelenterate luciferin, coelenterazine. Transient expression of aequorin in COS cells results in even greater abundance of luminescent protein. Aequorin protein is lost from digitonin-permeabilized cells to the same extent and at the same rate as lactate dehydrogenase (LDH), indicating cytosolic location of the indicator. Aequorin expressing cells treated with agonists of endogenous receptors were used in luminescence measurements to demonstrate that the reporter lines offer a highly sensitive and robust means of assaying changes in the concentration of cytosolic Ca2+ ion. Transient co-expression of the substance P receptor in aequorin reporter cells was also performed to demonstrate the feasibility of using this convenient and sensitive assay system for large scale screening of ligands that activate cell surface receptors coupled to increases in intracellular Ca2+.

Authors
Watkins NJ. Campbell AK.

Institution
Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff, U.K.

Title
Requirement of the C-terminal proline residue for stability of the Ca(2+)-activated photoprotein aequorin.

Source
Biochemical Journal. 293 ( Pt 1):181-5, 1993 Jul 1.

Abstract
cDNA coding for the Ca(2+)-activated photoprotein aequorin from the jellyfish Aequorea victoria has been engineered to investigate the role of the C-terminal proline residue in bioluminescence. Recombinant aequorin proteins were synthesized by PCR followed by in vitro transcription/translation, and characterized by specific activity, stability, and affinity for coelenterazine. The C-terminal proline residue of aequorin was shown to be essential for the long-term stability of the bound coelenterazine. Aequorin minus proline had only 1% of the specific activity of the wild-type after 2 h, and was virtually inactive after 18 h. The instability of this variant was further demonstrated by re-activating with a coelenterazine analogue (epsilon-coelenterazine), where maximum reactivation was reached in 15 min, and the luminescent activity was almost completely abolished within 3 h. Replacement of the C-terminal proline residue with histidine or glutamic acid decreased the specific activity to 10 and 19% of that of the wild-type respectively. However these variants were also unstable, having t1/2 values of 2.4 h and 2.3 h respectively. Enhancement of the Ca(2+)-independent light emission when proline was replaced by histidine confirmed the stabilizing role of the C-terminal proline. No significant effect of removal of the C-terminal proline was detected on the affinity for coelenterazine.

Authors
Shimomura O. Kishi Y. Inouye S.

Institution
Marine Biological Laboratory, Woods Hole, MA 02543.

Title
The relative rate of aequorin regeneration from apoaequorin and coelenterazine analogues.

Source
Biochemical Journal. 296 ( Pt 3):549-51, 1993 Dec 15.

Abstract
The regeneration of an active semi-synthetic aequorin, from apoaequorin produced in cells and a coelenterazine analogue, is a key step in measuring Ca2+ in the cells. The relative rates of the regeneration of semi-synthetic aequorins from apoaequorin and 28 synthetic coelenterazine analogues were compared. The results indicated that the rate is strongly influenced by the analogues used. The regeneration of ordinary aequorin with normal coelenterazine was relatively fast (50% regeneration in 22 min), whereas the rates of regenerating semi-synthetic aequorins with coelenterazine analogues varied widely, and all were slower than that of regenerating ordinary aequorin, except for e-type coelenterazines (containing an extra ethano group). The regeneration with e-type coelenterazines was significantly faster, indicating the possible superiority of e-type analogues in the intracellular regeneration of aequorin, especially when an increased sensitivity to Ca2+ is needed.

Authors
Sheu YA. Kricka LJ. Pritchett DB.

Institution
Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia.

Title
Measurement of intracellular calcium using bioluminescent aequorin expressed in human cells.

Source
Analytical Biochemistry. 209(2):343-7, 1993 Mar.

Abstract
Changes in intracellular free calcium concentration ([Ca2+]i) are involved in many important physiological responses. Detecting changes in [Ca2+]i is crucial to understanding the physiologic roles of intracellular free calcium. We have characterized changes of [Ca2+]i in human cells transfected with apoaequorin cDNA. When reconstituted in vivo by incubating transfected cells with coelenterazine, aequorin emits light upon binding free calcium and acts as a bioluminescent indicator for calcium. We have used this system to determine the concentration response relationship of serotonin for its receptor. Cells cotransfected with serotonin receptor cDNA and apoaequorin cDNA emitted light upon treatment with serotonin. The light emission responses were saturable and serotonin concentration-dependent, and they were inhibited by serotonin antagonists. Human 293 cells that stably express apoaequorin have been created. This system should facilitate the investigation of [Ca2+]i involvement in physiological and pathophysiological responses.

Authors
Knight MR. Read ND. Campbell AK. Trewavas AJ.

Institution
Institute of Cell and Molecular Biology, University of Edinburgh, United Kingdom.

Title
Imaging calcium dynamics in living plants using semi-synthetic recombinant aequorins.

Source
Journal of Cell Biology. 121(1):83-90, 1993 Apr.

Abstract
The genetic transformation of the higher plant Nicotiana plumbaginifolia to express the protein apoaequorin has recently been used as a method to measure cytosolic free calcium ([Ca2+]i) changes within intact living plants (Knight, M. R., A. K. Campbell, S. M. Smith, and A. J. Trewavas. 1991. Nature (Lond.). 352:524-526; Knight, M. R., S. M. Smith, and A. J. Trewavas. 1992. Proc. Natl. Acad. Sci. USA. 89:4967-4971). After treatment with the luminophore coelenterazine the calcium-activated photoprotein aequorin is formed within the cytosol of the cells of the transformed plants. Aequorin emits blue light in a dose-dependent manner upon binding free calcium (Ca2+). Thus the quantification of light emission from coelenterazine-treated transgenic plant cells provides a direct measurement of [Ca2+]i. In this paper, by using a highly sensitive photon-counting camera connected to a light microscope, we have for the first time imaged changes in [Ca2+]i in response to cold-shock, touch and wounding in different tissues of transgenic Nicotiana plants. Using this approach we have been able to observe tissue-specific [Ca2+]i responses. We also demonstrate how this method can be tailored by the use of different coelenterazine analogues which endow the resultant aequorin (termed semi-synthetic recombinant aeqorin) with different properties. By using h-coelenterazine, which renders the recombinant aequorin reporter more sensitive to Ca2+, we have been able to image relatively small changes in [Ca2+]i in response to touch and wounding: changes not detectable when standard coelenterazine is used. Reconstitution of recombinant aequorin with another coelenterazine analogue (e-coelenterazine) produces a semi-synthetic recombinant aequorin with a bimodal spectrum of luminescence emission. The ratio of luminescence at two wavelengths (421 and 477 nm) provides a simpler method for quantification of [Ca2+]i in vivo than was previously available. This approach has the benefit that no information is needed on the amount of expression, reconstitution or consumption of aequorin which is normally required for calibration with aequorin.

Authors
Lucas M. Solano F.

Institution
Departamento de Bioquimica y Biologia Molecular, Hospital Universitario Virgen Macarena, Facultad de Medicina, Sevilla, Spain.

Title
Coelenterazine is a superoxide anion-sensitive chemiluminescent probe: its usefulness in the assay of respiratory burst in neutrophils.

Source
Analytical Biochemistry. 206(2):273-7, 1992 Nov 1.

Abstract
The oxidation of free coelenterazine by superoxide anion was analyzed and compared to the oxidation by the semisynthetic photoprotein obelin, prepared by incorporation of synthetic coelenterazine into apoobelin. The oxidation of bound coelenterazine was triggered upon binding of calcium to the reconstituted photoprotein. The oxidation of free synthetic coelenterazine, in the absence of the apoprotein, was triggered by superoxide anion. The production of reactive oxygen metabolites by fMet-Leu-Phe- and 4b-phorbol 12b-myristate 13a-acetate-stimulated neutrophils was studied by means of the luminescence of synthetic coelenterazine. The features of this chemiluminescent probe were compared with those of luminol and are summarized as follows: (a) coelenterazine-dependent chemiluminescence was inhibited by superoxide dismutase; (b) coelenterazine was as sensitive as luminol in detecting the oxidative burst of neutrophils; (c) azide failed to inhibit coelenterazine chemiluminescence; (d) in contrast with luminol, which requires the catalytic removal of hydrogen peroxide, coelenterazine chemiluminescence did not depend on the activity of cell-derived myeloperoxidase. These results indicate the usefulness of coelenterazine as a very sensitive and specific chemiluminescence probe of superoxide anion.

Authors
Rizzuto R. Simpson AW. Brini M. Pozzan T.

Institution
Department of Biomedical Sciences, University of Padova, Italy.

Title
Rapid changes of mitochondrial Ca2+ revealed by specifically targeted recombinant aequorin [published erratum appears in Nature 1992 Dec 24-31;360(6406):768].

Source
Nature. 358(6384):325-7, 1992 Jul 23.

Abstract
Introduction of Ca2+ indicators (photoproteins, fluorescent dyes) that can be trapped in the cytosolic compartment of living cells has yielded major advances in our knowledge of Ca2+ homeostasis. Ca2+ however regulates functions not only in the cytosol but also within various organelles where indicators have not yet been specifically targeted. Here we present a novel procedure by which the free Ca2+ concentration of mitochondria, [Ca2+]m, can be monitored continuously at rest and during stimulation. The complementary DNA for the Ca2+ sensitive photoprotein aequorin was fused in frame with that encoding a mitochondrial presequence. The hybrid cDNA was transfected into bovine endothelial cells and stable clones were obtained expressing variable amounts of mitochondrially targeted apoaequorin. The functional photoprotein could be reconstituted in intact cells by incubation with purified coelenterazine and [Ca2+]m could thus be monitored in situ. This allowed the unprecedented direct demonstration that agonist-stimulated elevations of cytosolic free Ca2+, [Ca2+]i, (measured in parallel with Fura-2) evoke rapid and transient increases of [Ca2+]m, which can be prevented by pretreatment with a mitochondrial uncoupler. The possibility of targeting aequorin to cellular organelles not only offers a new and powerful method for studying aspects of Ca2+ homeostasis that up to now could not be directly approached, but might also be used in the future as a tool to report in situ a variety of apparently unrelated phenomena of wide biological interest.

Authors
Inouye S. Tsuji FI.

Institution
Yokohama Research Center, Chisso Corporation, Kanagawa, Japan.

Title
Monitoring gene expression in Chinese hamster ovary cells using secreted apoaequorin.

Source
Analytical Biochemistry. 201(1):114-8, 1992 Feb 14.

Abstract
A luminescence method for monitoring gene expression in Chinese hamster ovary cells using apoaequorin as a secreted reporter enzyme is described. In this method, the cell is not disrupted prior to assay as in the earlier aequorin procedure and in the firefly method. The apoaequorin secretion vector is constructed by fusing the DNA fragment of the signal peptide sequence of human follistatin to the apoaequorin gene. Transfection of Chinese hamster ovary cells with the vector causes the apoaequorin to be secreted directly into the culture medium. Assay is carried out by removing a small aliquot of the culture medium, incubating it with coelenterazine, and adding Ca2+ to trigger light emission from the regenerated aequorin. The light intensity is measured with a photomultiplier photometer and is proportional to the amount of apoaequorin present. The method is highly specific and sensitive and can be carried out in a relatively short period of time.

Authors
Lorenz WW. McCann RO. Longiaru M. Cormier MJ.

Institution
Department of Biochemistry, University of Georgia, Athens 30602.

Title
Isolation and expression of a cDNA encoding Renilla reniformis luciferase.

Source
Proceedings of the National Academy of Sciences of the United States of America. 88(10):4438-42, 1991 May 15.

Abstract
Renilla reniformis is an anthozoan coelenterate capable of exhibiting bioluminescence. Bioluminescence in Renilla results from the oxidation of coelenterate luciferin (coelenterazine) by luciferase [Renilla-luciferin:oxygen 2-oxidoreductase (decarboxylating), EC 1.13.12.5]. In vivo, the excited state luciferin-luciferase complex undergoes the process of nonradiative energy transfer to an accessory protein, green fluorescent protein, which results in green bioluminescence. In vitro, Renilla luciferase emits blue light in the absence of any green fluorescent protein. A Renilla cDNA library has been constructed in lambda gt11 and screened by plaque hybridization with two oligonucleotide probes. We report here the isolation and characterization of a luciferase cDNA and its gene product. The recombinant luciferase expressed in Escherichia coli is identical to native luciferase as determined by SDS/PAGE, immunoblot analysis, and bioluminescence emission characteristics.

Authors
Knight MR. Campbell AK. Smith SM. Trewavas AJ.

Institution
Institute of Cell and Molecular Biology, University of Edinburgh, UK.

Title
Transgenic plant aequorin reports the effects of touch and cold-shock and elicitors on cytoplasmic calcium.

Source
Nature. 352(6335):524-6, 1991 Aug 8.

Abstract
Methods for measuring plant cytoplasmic calcium using microelectrodes or microinjected fluorescent dyes are associated with extensive technical problems, so measurements have been limited to single or small groups of cells in tissue strips or protoplasts. Aequorin is a calcium-sensitive luminescent protein from the coelenterate Aequorea victoria (A. forskalea) which is formed from apoaequorin, a polypeptide of relative molecular mass approximately 22,000, and coelenterazine, a hydrophobic luminophore. Microinjected aequorin has been widely used for intracellular calcium measurement in animal cells, but its use in plants has been limited to exceptionally large cells. We show here that aequorin can be reconstituted in transformed plants and that it reports calcium changes induced by touch, cold-shock and fungal elicitors. Reconstituted aequorin is cytoplasmic and nonperturbing; measurements can be made on whole plants and a calcium indicator can be constituted in every viable cell. Now that apoaequorin can be targeted to specific organelles, cells and tissues, with the range of coelenterazines with differing calcium sensitivities and properties available, this new method could be valuable for determining the role of calcium in intracellular signalling processes in plants.

Authors
Nakajima-Shimada J. Iida H. Tsuji FI. Anraku Y.

Institution
Division of Cell Proliferation, National Institute for Basic Biology, Okazaki, Japan.

Title
Monitoring of intracellular calcium in Saccharomyces cerevisiae with an apoaequorin cDNA expression system.

Source
Proceedings of the National Academy of Sciences of the United States of America. 88(15):6878-82, 1991 Aug 1.

Abstract
A method is described for measuring cytosolic free Ca2+ and its time-dependent changes in the yeast Saccharomyces cerevisiae by using the luminescent protein aequorin as a Ca(2+)-specific indicator. This method with intact yeast cells is labeled "in vivo" to distinguish it from methods with cell extracts, labeled "in vitro." A plasmid in which the apoaequorin cDNA was joined downstream from the glyceraldehyde-3-phosphate dehydrogenase gene promoter was constructed and introduced into yeast cells. The intracellular concentration of apoaequorin expressed by the cDNA was approximately 1 microM, which was high enough to detect the cytosolic Ca2+. Growth of the transformed cells was normal. In the in vitro method, apoaequorin in crude cell extracts was regenerated into aequorin by mixing with coelenterazine, the substrate for the luminescence reaction, whereas in the in vivo method, aequorin was regenerated by incubating intact cells with coelenterazine. Simultaneous addition of 10 mM CaCl2 and 10 microM A23187, a Ca2+ ionophore, to coelenterazine-incorporated cells generated luminescence. Coelenterazine-incorporated cells also responded to native extracellular stimuli. A mating pheromone, alpha-factor, added to cells of mating type a or alpha, generated extracellular Ca(2+)-dependent luminescence specifically in a mating type cells, with maximal intensity occurring 45-50 min after addition of alpha-factor. Glucose added to glucose-starved G0/G1 cells stimulated an increase in extracellular Ca(2+)-dependent luminescence with maximal intensity occurring 2 min after addition. These results show the usefulness of the aequorin system in monitoring [Ca2+]i response to extracellular stimuli in yeast cells.

Authors
Shimomura O. Inouye S. Musicki B. Kishi Y.

Institution
Marine Biological Laboratory, Woods Hole, MA 02543.

Title
Recombinant aequorin and recombinant semi-synthetic aequorins. Cellular Ca2+ ion indicators.

Source
Biochemical Journal. 270(2):309-12, 1990 Sep 1.

Abstract
Properties of a recombinant aequorin were investigated in comparison with those of natural aequorin. In chromatographic behaviour the recombinant aequorin did not match any of ten isoaequorins tested, although it was very similar to aequorin J. Its sensitivity to Ca2+ was found to be higher than that of any isoaequorin except aequorin D. The recombinant aequorin exhibited no toxicity when tested in various kinds of cells, even where samples of natural aequorin had been found to be toxic. Properties of four recombinant semi-synthetic aequorins (fch-, hcp-, e- and n-types), prepared from the recombinant apo-aequorin and synthetic analogues of coelenterazine, were approximately parallel with those of corresponding semi-synthetic aequorins prepared from natural apo-aequorin. Both recombinant e-aequorin and natural e-aequorin J luminesced with high values of the luminescence intensity ratio I400/I465, although the ratios were not pCa-dependent. The recombinant aequorin and recombinant semi-synthetic aequorins are highly suited for monitoring cellular Ca2+.

Authors
Muller T. Campbell AK.

Institution
Department of Biochemistry, University of Fribourg, Switzerland.

Title
The chromophore of pholasin: a highly luminescent protein.

Source
Journal of Bioluminescence & Chemiluminescence. 5(1):25-30, 1990 Jan-Mar.

Abstract
Pholasin is the photoprotein extracted from the marine bivalve Pholas dactylus. It undergoes an oxidative chemiluminescent reaction to oxypholasin with superoxide anion, hypochlorite, peroxidases and other oxidants. Since the observed absorbance and chemiluminescent emission spectra of pholasin solutions cannot be brought about solely by the amino acids composing the protein, there has to be a chemiluminescent chromophore. However, little is known about the chemical nature of this molecule. This work seeks to identify the chemical structure of the luminescent prosthetic group of pholasin. Pholasin could not be reactivated using chromophores from the hydroid Obelia geniculata (coelenterazine) and from the ostracod shrimp Vargula (formerly Cypridina) hilgendorfi. Furthermore, the reaction product of the Vargula chromophore could not be detected in solutions containing oxypholasin. Fluorescence analysis of such a solution revealed a compound with an emission spectrum (lambda max 480 nm; excitation at 320 nm), resembling the emission spectrum of the chemiluminescent reaction. This fluorescent substance was separated by gel filtration. It exhibited an apparent molecular mass of less than 2000. Fluorescence measurements of extracts of partially purified pholasin suggested that a flavin moiety may be involved in pholasin luminescence.

Authors
Watanabe M. Kurihara S. Inouye S. Ohno T.

Institution
1st Department of Microbiology, Jikei University School of Medicine, Tokyo, Japan.

Title
Binding of murine monoclonal antibodies to the active and inactive configurations of aequorin.

Source
FEBS Letters. 246(1-2):73-7, 1989 Mar 27.

Abstract
Murine monoclonal IgG1 antibodies (MAb), designated Aq-11 and Aq-12, were prepared against the photoprotein aequorin from jelly fish. Aequorin is a calcium-sensitive photoprotein which consists of a single polypeptide chain, apoaequorin, and a functional chromophore, coelenterazine. Native aequorin consists of two species with molecular masses of 25 and 23.5 kDa. MAb Aq-12 was found by immunoblot analysis to bind specifically to the 25 kDa species, while MAb Aq-11 reacted with the 23.5 kDa protein. Activation of apoaequorin with coelenterazine was associated with a shift of the 23.5 kDa molecule to the 25 kDa species. In contrast, treatment with calcium ions induced a shift back to the 23.5 kDa form. These changes between the active and inactive forms were identified by reactivity with MAbs Aq-11 and Aq-12. The results thus indicate that these MAbs should be useful in monitoring activation of this photoprotein.

Authors
Inouye S. Aoyama S. Miyata T. Tsuji FI. Sakaki Y.

Institution
Basic Research Laboratory, Chisso Corporation, Kanagawa.

Title
Overexpression and purification of the recombinant Ca2+-binding protein, apoaequorin.

Source
Journal of Biochemistry. 105(3):473-7, 1989 Mar.

Abstract
The small, monomeric Ca2+-binding photoprotein, aequorin, emits blue light by an intramolecular reaction when mixed with Ca2+. The photoprotein is made up of coelenterazine and molecular oxygen, bound noncovalently to apoaequorin (apoprotein). The chemical steps leading to light emission, involving the oxidative degradation of coelenterazine, have been studied extensively, but little is known about the active site and how the molecule catalyzes the oxidation of coelenterazine. The three-dimensional structure of the protein has not been determined and therefore answers to these questions have remained unavailable. The present paper describes a procedure for preparing fairly large amounts of apoaequorin and aequorin for X-ray crystallographic studies. It consists of fusing the apoaequorin cDNA to the signal peptide coding sequence of the outer membrane protein A of Escherichia coli, which is under the control of the lipoprotein promoter. When the cDNA was expressed in E. coli, a large excess of the recombinant protein was produced and released into the culture medium. Purification of the protein was accomplished by acid precipitation and DEAE-cellulose chromatography. The procedure yielded 7.4 mg of recombinant apoaequorin with a purity greater than 95% from 200 ml of culture medium. On regeneration with coelenterazine, the recombinant aequorin was fully active with Ca2+.

Authors
Kurose K. Inouye S. Sakaki Y. Tsuji FI.

Institution
Research Laboratory for Genetic Information, Kyushu University, Fukuoka, Japan.

Title
Bioluminescence of the Ca2+-binding photoprotein aequorin after cysteine modification.

Source
Proceedings of the National Academy of Sciences of the United States of America. 86(1):80-4, 1989 Jan.

Abstract
Aequorin is a monomeric Ca2+-binding protein (Mr, 21,400) that emits light upon reacting with Ca2+. The protein has three Ca2+-binding sites, three cysteine residues, and a noncovalently bound chromophore that consists of coelenterazine and molecular oxygen. Light is emitted via an intramolecular reaction in which coelenterazine is oxidized by the bound oxygen. After light emission, aequorin may be regenerated by incubating the protein with coelenterazine, dissolved oxygen, EDTA, and 2-mercaptoethanol. To understand structure-function relationships in this protein, we used the technique of site-specific mutagenesis to replace the three cysteine residues with serine. Six of the seven modified aequorins had reduced luminescence activity, whereas the seventh with all three cysteines replaced by serine had luminescence activity equal to or greater than that of the wild-type aequorin. Further, the time required for the regeneration of the triply substituted aequorin was substantially increased compared to the time required for the regeneration of the wild-type aequorin. The results suggest that cysteine plays an important role in the regeneration of aequorin but not in its catalytic activity.

Authors
Shimomura O. Musicki B. Kishi Y.

Institution
Marine Biological Laboratory, Woods Hole, MA 02543.

Title
Semi-synthetic aequorins with improved sensitivity to Ca2+ ions.

Source
Biochemical Journal. 261(3):913-20, 1989 Aug 1.

Abstract
Thirty-seven coelenterazine analogues were synthesized and incorporated into apo-aequorin, yielding 30 semi-synthetic aequorins that have the capacity to emit a significant amount of light in the presence of Ca2+. The properties of resultant photoproteins were investigated. The most prominent feature of those photoproteins was the wide range in their sensitivities to Ca2+ concentration. The relative intensity of Ca2+-triggered luminescence of the photoproteins ranged from 0.01 to 190 when compared with natural aequorin (relative intensity 1.0) at pCa 6 for the cases where the relative intensity is less than 1 and at pCa 7 for the cases where the relative intensity is higher than 1. Eight of the semi-synthetic aequorins belonged to the class of e-aequorin. With two of those photoproteins, the degree of dependence of the luminescence intensity ratio I400/I465 on pCa was greater than that with e-aequorin, suggesting that these two photoproteins are possibly superior to e-aequorin in measuring Ca2+ concentration by the ratio method.

Authors
Casadei J. Powell MJ. Kenten JH.

Institution
IGEN Inc., Rockville, MD 20852.

Title
Characterization of a chimeric aequorin molecule expressed in myeloma cells.

Source
Journal of Bioluminescence & Chemiluminescence. 4(1):346-50, 1989 Jul.

Abstract
We have constructed a chimeric aequorin consisting of a fragment of the anti-NP immunoglobulin gene fused to the aequorin gene. Expression in a myeloma cell line has produced a Fab'-like molecule that has the ability to bind NIP specifically and generate bioluminescent activity. It takes approximately 8 h at 4 degrees C in the presence of 2-mercaptoethanol and coelenterazine to regenerate luminescent activity. While the flash kinetics of this recombinant molecule are similar to native aequorin, its quantum efficiency is ten times lower. Preliminary studies have been conducted to ascertain its usefulness for immunoassays. We have shown for this chimeric aequorin 7 x 10(-19) moles can be detected in solution, also it can be used in a solid-phase assay and is stably stored at -70 degrees C for at least 2 months.

Authors
Campbell AK. Patel AK. Razavi ZS. McCapra F.

Institution
Department of Medical Biochemistry, University of Wales College of Medicine, Cardiff, U.K.

Title
Formation of the Ca2+-activated photoprotein obelin from apo-obelin and mRNA inside human neutrophils.

Source
Biochemical Journal. 252(1):143-9, 1988 May 15.

Abstract
1. A method has been developed to incorporate the apoprotein of the Ca2+-activated photoprotein obelin, and mRNA purified from the hydroid Obelia, into the cytoplasm of intact human neutrophils. This was based on internal release from pH-sensitive immunoliposomes taken up initially by phagocytosis. 2. Addition of the prosthetic group of obelin, coelenterazine, to these cells containing apo-obelin or Obelia mRNA resulted in formation of active Ca2+-activated obelin. 3. The obelin formed within the neutrophils responded to the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (1 microM) and to the membrane attack complex of complement (C5B6789n). 4. The formation of the apo-obelin from mRNA within neutrophils was inhibited by over 80% in the absence of added amino acids, and by over 90% by the protein-synthesis inhibitor puromycin (100 micrograms/ml). 5. The translation of Obelia mRNA inside cells provides a method for circumventing consumption of Ca2+-activated photoproteins during cell activation or injury, and for monitoring protein synthesis in living cells.

Authors
Shimomura O. Musicki B. Kishi Y.

Institution
Marine Biological Laboratory, Woods Hole, MA 02543.

Title
Semi-synthetic aequorin. An improved tool for the measurement of calcium ion concentration.

Source
Biochemical Journal. 251(2):405-10, 1988 Apr 15.

Abstract
The photoprotein aequorin isolated from the jellyfish Aequorea emits blue light in the presence of Ca2+ by an intramolecular process that involves chemical transformation of the coelenterazine moiety into coelenteramide and CO2. Because of its high sensitivity to Ca2+, aequorin has widely been used as a Ca2+ indicator in various biological systems. We have replaced the coelenterazine moiety in the protein with several synthetic coelenterazine analogues, providing semi-synthetic Ca2+-sensitive photoproteins. One of the semi-synthetic photoproteins, derived from coelenterazine analogue (II) (with an extra ethano group), showed highly promising properties for the measurement of Ca2+, namely (1) the rise time of luminescence in response to Ca2+ was shortened by approx. 4-fold compared with native aequorin and (2) the luminescence spectrum showed two peaks at 405 nm and 465 nm and the ratio of their peak heights was dependent on Ca2+ concentration in the range of pCa 5-7, thus allowing the determination of [Ca2+] directly from the ratio of two peak intensities. Coelenterazine analogue (I) (with a hydroxy group replaced by an amino group) was also incorporated into apo-aequorin, yielding a Ca2+-sensitive photoprotein, which indicates that an electrostatic interaction between the phenolate group in the coelenterazine moiety and some cationic centre in apo-aequorin is not important in native aequorin, contrary to a previous suggestion.

Authors
Shimomura O.

Title
Bioluminescence in the sea: photoprotein systems. [Review]

Source
Symposia of the Society for Experimental Biology. 39:351-72, 1985.

Abstract
Photoproteins are the primary reactants of the light-emitting reactions of various bioluminescent organisms. A photoprotein emits light in proportion to its amount, like a luciferin, but its light-emitting reaction does not require a luciferase. There are about two dozen types of bioluminescent organisms for which substantial biochemical knowledge is presently available, and about one third of them involve photoproteins. Most photoproteins are found in marine organisms. There are various types of photoproteins: the photoproteins of coelenterates, ctenophores and radiolarians require Ca2+ to trigger their luminescence; the photoproteins of the bivalve Pholas and of the scale worm appear to involve superoxide radicals and O2 in their light-emitting reactions; the photoprotein of euphausiid shrimps emits light only in the presence of a special fluorescent compound; the photoprotein of the millipede Luminodesmus, the only known example of terrestrial origin, requires ATP and Mg2+ to emit light. The Ca2+-sensitive photoproteins of coelenterates have been most frequently studied and most widely used. Therefore, they are overwhelmingly popular compared with other types. All coelenterate photoproteins, including aequorin, halistaurin, obelin and phialidin, have relative molecular masses close to 20 000, contain an identical functional group, and emit blue light in aqueous solution when a trace of Ca2+ is added, in the presence or absence of molecular oxygen. Aequorin contains an oxygenated form of coelenterazine in its functional group. When Ca2+ is added, aequorin decomposes into three parts, i.e., apo-aequorin, coelenteramide and CO2, accompanied by the emission of light. Apo-aequorin can be reconstituted into active aequorin indistinguishable from the original sample, by incubation with an excess of coelenterazine in a buffer containing 5 mM-EDTA and a trace of 2-mercaptoethanol, even at 0 degree C. Thus, aequorin and other coelenterate photoproteins can be luminesced and recharged repeatedly. The regeneration of coelenterate photoproteins in this manner probably takes place in vivo, utilizing stored coelenterazine. The photoproteins of coelenterates, and their chemically modified forms, are useful in measuring and monitoring calcium ions in biological systems, especially in single cells. [References: 66]

Authors
Inouye S. Noguchi M. Sakaki Y. Takagi Y. Miyata T. Iwanaga S. Miyata T. Tsuji FI.

Title
Cloning and sequence analysis of cDNA for the luminescent protein aequorin.

Source
Proceedings of the National Academy of Sciences of the United States of America. 82(10):3154-8, 1985 May.

Abstract
The luminescent jellyfish Aequorea contains a photoprotein, aequorin, which emits light by an intramolecular reaction in the presence of a trace amount of Ca2+. A cDNA library of Aequorea was constructed and clones carrying the cDNA for the Ca2+-dependent photoprotein were isolated by the method of colony hybridization using synthetic oligonucleotide probes. The primary structure of the protein deduced from the nucleotide sequence showed that the protein is composed of 189 amino acid residues and has three E-F hand structures that are characteristic for Ca2+-binding sites. The sequence also suggested that the protein has hydrophobic regions at which the protein may interact with its functional chromophore, coelenterazine.

Authors
Anctil M. Shimomura O.

Title
Mechanism of photoinactivation and re-activation in the bioluminescence system of the ctenophore Mnemiopsis.

Source
Biochemical Journal. 221(1):269-72, 1984 Jul 1.

Abstract
The bioluminescence of the ctenophore Mnemiopsis takes place when the photoprotein mnemiopsin in the photocytes reacts with Ca2+. The luminescence is inhibited in sunlight and this photoinhibition is reversible by keeping the live specimens in the dark. The extracts of mnemiopsin are similarly photoinhibited, but the photoinhibition cannot be reversed in the dark. We have found that photo-inhibited mnemiopsin can be re-activated in the dark by incubation with coelenterazine and O2 only in solutions having a pH very close to 9.0. The re-activation in vivo probably takes place in the same manner, using the coelenterazine that is supplied from its abundant storage form. Various lines of experimental evidence suggest that the photoinactivation of mnemiopsin results in the dissociation of coelenterazine and oxygen from the molecule of photoprotein; the dissociated form of the former molecule is an inactive form of coelenterazine, not free coelenterazine.

Authors
Shimomura O. Masugi T. Johnson FH. Haneda Y.

Title
Properties and reaction mechanism of the bioluminescence system of the deep-sea shrimp Oplophorus gracilorostris.

Source
Biochemistry. 17(6):994-8, 1978 Mar 21.

Abstract
The bioluminescent reaction of Oplophorus takes place when the oxidation of coelenterazine (the luciferin) with molecular oxygen is catalyzed by Oplophorus luciferase, resulting in light of maximum intensity at 462 nm and the products CO2 and coelenteramide. Oplophorus luciferase has now been obtained in a highly purified state. Optimum luminescence occurs at pH 9 in the presence of 0.05--0.1 M NaCl at 40 degrees C, and, due to the unusual resistance of this enzyme to heat, visible luminescence occurs at temperatures above 70 degrees C when partially purified enzyme is used. The specific activity of purest preparations is 1.75 X 10(15) photons s-1 mg-1 at 23 degrees C. At pH 8.7, native luciferase has a molecular weight of approximately 130 000, apparently comprising 4 monomers of 31 000; at lower pHs, the native luciferase tends to polymerize. The quantum yield of coelenterazine is 0.34 at 22 degrees C with this enzyme. After the luminescent reaction, the spent solution is nonfluorescent, and likewise solutions of luciferase alone. When the bioluminescent reaction was carried out in the presence of 18O2, the product CO2 contained more than 50% C18O16O, supporting the dioxetane mechanism, but without ruling out the linear peroxide mechanism.

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